2.3. DNA extraction, sequencing, and alignment

DNA was extracted using DNeasy Blood and Tissue Kits (Qiagen) following the manufacturer's protocol with slight modifications. All samples were lysed overnight in a thermomixer at 55°C between steps 2 and 3 in the manufacturer's protocol. At step 3, the lysis buffer (AL) was heated to 50°C before addition, and in step 7, the elution buffer (AE) was warmed to 70°C before addition. After pipetting buffer AE onto the spin column filter, the columns were incubated for up to 15 min at room temperature. The final elution was done twice into the same collection tube, leading to a total extract volume of 100 μl. Extract DNA concentrations were measured with a Qubit fluorometer using the Qubit 1X dsDNA HS Assay Kit (Invitrogen) following the manufacturer's protocol.

The standard barcode of the mitochondrial COI gene was PCR amplified with the universal primers LCO1490 and HCO2198 (Folmer et al., 1994). PCR reactions were carried out in volumes of 25 μl, including 2 μl template DNA, 0.5 μM of each primer, 1 U of Taq polymerase (Invitrogen), 0.2 μM of each dNTP, 1X Mg‐free PCR buffer, and 1.5 μM MgCl₂. Thermal cycling conditions included initial denaturation at 94°C for 3 min, followed by 30 cycles of 94°C for 45 s, 50°C for 30 s, 72°C for 90 s, and a final extension at 72°C for 10 min. PCR products were checked through electrophoresis on 1.5% agarose gels stained with ethidium bromide. Whenever multiple bands were present in the gels, we performed a new PCR reaction with Q5 High‐Fidelity 2X Mastermix (New England BioLabs Inc.), in a total reaction volume of 25 μl, including 2 μl template DNA and 0.5 μM of each primer. These reactions were run with an initial denaturation at 98°C for 30 s, then 30 cycles of 98°C for 10 s, 50°C for 30 s, and 72°C for 30 s, followed by a final extension step at 72°C for 2 min.

Successfully amplified products were purified enzymatically from unincorporated nucleotides and primers before sequencing. For this, 15 μl of PCR product was mixed with 30 U Exonuclease I and 3 U FastAP Thermosensitive Alkaline Phosphatase (PCR cleanup prior to sequencing, Thermo Scientific), then incubated at 37°C for 15 min, followed by 85°C for 15 min to stop the reaction. The products were Sanger sequenced in both directions using the amplification primers at Macrogen Inc., The Netherlands.

Resultant sequences were edited and aligned using Geneious Prime 2020.1 software (Biomatters Ltd). Final sample sequences were aligned with each other using the MAFFT multiple sequence alignment algorithm (Katoh & Standley, 2013) implemented within Geneious. We constructed two different barcode sequence alignments for the subsequent analyses: (i) an alignment including only the 132 samples analyzed in this study; and (ii) an expanded alignment including a further 66 reference sequences retrieved from the GenBank and BOLD databases (metadata S1 and data S1 in Nyman et al., 2022). The reference barcode sequences for the latter dataset were selected to represent (i) identified reference individuals that constituted close hits for our own barcodes; (ii) identified specimens of species that our literature review indicated as parasitoids of the focal geometrids, but that were not obtained from our own rearings; and (iii) representative congeners of parasitoids that are known to parasitize the focal moth species. The last class also included five species that have been listed as parasitoids of the focal moth species, but that we consider doubtful or unlikely associates (Table S1).